Biological samples are commonly preserved to support a broad variety of biomedical and biological research that includes but is not limited to translational research, molecular medicine, and biomarker discovery. Biological samples include any samples which are of animal (including human), plant, protozoal, fungal, bacterial, viral, or other biological origin. For example, biological samples include, but are not limited to, organisms and/or biological fluids isolated from or excreted by an organism such as plasma, serum, urine, whole blood, cord blood, other blood-based derivatives, cerebral spinal fluid, mucus (from respiratory tract, cervical), ascites, saliva, amniotic fluid, seminal fluid, tears, sweat, any fluids from plants (including sap); cells (e.g., animal, plant, protozoal, fungal, or bacterial cells, including buffy coat cells; cell lysates, homogenates, or suspensions; microsomes; cellular organelles (e.g., mitochondria); nucleic acids (e.g., RNA, DNA), including chromosomal DNA, mitochondrial DNA, and plasmids (e.g., seed plasmids); small molecule compounds in suspension or solution (e.g. small molecule compounds in DMSO); and other fluid-based biological samples. Biological samples may also include plants, portions of plants (e.g., seeds) and tissues (e.g., muscle, fat, skin, etc.), including healthy tissue and diseased tissue (e.g., tumors).
Biobanks typically store these valuable samples in containers (e.g., tubes, vials, or the like) and cryopreserve them (e.g., in freezers at −80 degrees centigrade, or lower using liquid Nitrogen or the vapor phase above liquid Nitrogen) to preserve the biochemical composition and integrity of the frozen sample as close as possible to the in vivo state to facilitate accurate, reproducible analyses of the samples.
From time to time, it may be desirable to run one or more tests on a sample that has been frozen. For example, a researcher may want to perform tests on a set of samples having certain characteristics. A particular sample may contain enough material to support a number of different tests. In order to conserve resources, smaller samples known as aliquots are commonly taken from larger cryopreserved samples (which are sometimes referred to as parent samples) for use in one or more tests so the remainder of the parent sample will be available for one or more different future tests.
Biobanks have adopted different ways to address this need to provide sample aliquots. One option is to freeze a sample in large volume, thaw it when aliquots are requested and then refreeze any remainder of the parent sample for storage in the cryopreserved state until future aliquots are needed. This option makes efficient use of frozen storage space; yet this efficiency comes at the cost of sample quality. Exposing a sample repeatedly to freeze/thaw cycles can degrade the sample's critical biological molecules (e.g., RNA) and damage biomarkers, either of which could compromise the results of any study using data obtained from the damaged samples.
Another option is to freeze a sample in large volume, thaw it when an aliquot is requested, subdivide the remainder of the parent sample in small volumes to make additional aliquots for future tests and then refreeze these smaller volume aliquots to cryopreserve each aliquot separately until needed for a future test. This approach limits the number of freeze/thaw cycles to which a sample is exposed, but there is added expense associated with the larger volume of frozen storage space, labor, and larger inventory of sample containers (e.g. tubes, vials, or the like) required to maintain the cryopreserved aliquots. Moreover, the aliquots can be degraded or damaged by even a limited number freeze/thaw cycles.
Yet another approach is to divide a large volume sample into smaller volume aliquots before freezing them for the first time. This approach can limit the number of freeze thaw cycles to which a sample may be subjected to only one; yet, there are disadvantages associated with the costs of labor, frozen storage space, and sample container inventory requirements with this approach.
When aliquotting using any of the above approaches, the sampling devices used to make the aliquots must be thoroughly cleaned before being used again on another sample. In some cases all traces of the sample may not be removed during the cleaning process (e.g., due to human error, such as failure to supply a cleaning station with the proper cleaning fluids or the like). Contamination of the samples can negatively affect the viability and integrity of a sample. In other cases, a user may wish to use a new sampling device for every sample when a high level of cleanliness is required. For example, some applications (e.g., cell-based material research, forensic analysis, etc.) require use of equipment that is substantially free from nucleotides, nucleic acids (e.g., DNA and RNA), nucleases (e.g., DNase and RNase), and any other enzymes or biological molecules that can degrade or contaminate the biological sample. Other applications may require sterile working conditions or equipment. Although it is possible to clean a sampling device sufficiently to achieve these high levels of cleanliness as part of a reliable workflow, some users may feel more confident if the sampling device is not re-used.
U.S. Pat. No. 8,448,456, the contents of which are hereby incorporated by reference, discloses a system for extracting frozen sample cores from a frozen biological sample without thawing the original (parent) sample. The system uses a drill including a hollow coring bit to take frozen core samples from the original parent samples without thawing the parent samples. One or more frozen sample cores from a parent sample, depending on the amount of sample needed for a particular test, can constitute the aliquot for the test. After an aliquot is obtained from a parent sample, the remainder of the sample is returned to frozen storage in its original container until another aliquot from the parent sample is needed for a future test.
U.S. application Ser. No. 13/359,301, the contents of which are hereby incorporated by reference, discloses a robotic end effector for collecting frozen aliquots from an array of frozen samples in a plurality of containers. The end effector uses a hollow coring bit to take frozen sample cores from the original samples without thawing the parent samples. A fill-level detection system detects the position of the surfaces of the frozen samples to determine if a sufficient amount of frozen sample cores have been taken from a particular frozen sample to obtain a predetermined amount of material from that frozen sample.
PCT application No. PCT/US2011/61214 and U.S. provisional application No. 61/418,688, the contents of which are hereby incorporated by reference, disclose a method of obtaining an aliquot of a frozen sample using a coring device. The location of the coring is selected to be at a radial position where the concentration of a substance of interest in the frozen sample core is representative of the overall concentration of the substance in the parent sample.
U.S. Provisional Application No. 61/640,662 and U.S. application Ser. No. 13/489,234, the contents of which are hereby incorporated by reference, disclose a machine vision system for use with a system for obtaining frozen sample cores. The machine vision system includes a camera and a processor that receives image data from the camera to determine locations where frozen sample cores have already been taken from a frozen biological sample.
In pathology and biomedical research, tissue samples are often stored and sampled. Conventionally, the tissue samples were subjected to formalin fixation and embedded in paraffin or optimal cutting temperature compound (OCT). The embedded tissue is fixed to a slide sectioning device, such as a microtome or cryotome, and a thin section of the tissue is sliced off the top of the sample. The thin section is evaluated on a slide, and the area of interest of the sample (e.g., tumor) is identified (e.g., using a marking device to circle the area of interest). The slide is then lined up with the remainder of the tissue sample to determine where the area of interest is on the remaining tissue. The tissue sample is then moved to a processing or sampling area or device, and a sample is taken from the area of interest, typically by using a scalpel to cut the sample into pieces and extract a portion of tissue from the area of interest. One problem with formalin fixed embedded tissue is that biomarkers degrade and the research quality of the tissue is negatively affected by the fixation process. Thus, the use of frozen tissue is desirable over fixed material. However, the frozen tissue samples are typically stored in a variety of containers and processed with methods that require thawing of the samples to obtain portions of tissue from the areas of interest. The frozen tissue samples must still be sectioned for a slide and then moved to a sampling device. The variety of containers used to store a tissue sample, as well as the multiple apparatuses and fixtures that are needed to determine the area of interest and to sample a tissue sample, complicate the process.
The present inventors have developed systems and methods, which will be described below, that improve the ability to provide frozen aliquots from a frozen biological sample (e.g., frozen fluid and/or frozen tissue samples) using a system that extracts frozen sample cores from frozen biological samples without thawing the original (parent) samples. Furthermore, the present inventors have developed systems and methods to reduce the complexity of frozen tissue sample sampling and create a uniform process by mounting the frozen tissue sample in a tissue sample container that can be used with a slide sectioning device and with a frozen tissue sampling device.